RNA is an ancient carrier of biological information, whose many functions became necessary for the evolution of cellular life, and transcriptional and post-transcriptional regulation of RNA is central to health and the progression of human disease. Remarkably, we have discovered that thousands of human genes produce circular RNA (circRNA) isoforms and in hundreds of genes, the circRNA is more abundant than the linear isoform. Our recent work has shown that circRNA expression is particularly regulated during human development. We have also demonstrated that circular RNAs are produced in organisms separated by billions of years of evolution, which suggests that the machinery, and by implication, function, of circRNA is central to eukaryotic gene expression programs: either conserved over billions of years, or a feature that has re-evolved multiple times which implies a functional role for circRNAs in the cell. Together, our work suggests a fundamental hypothesis that alternative splicing has functional consequences apart from protein production, including the production of circRNA isoforms. Yet, the field lacks a predictive mechanistic model of the cis sequences and trans-acting factors that specifically regulate circRNA, meaning a) the biochemical signaling pathways used by the cell to produce circRNA are unknown; b) we lack molecular tools to specifically express circRNA without background transcription of off-target RNAs. Such tools are required for discovery and rigorous experimental tests of circRNA function. This proposal aims to discover the mechanisms controlling circRNA production and regulation and promises to reveal novel biology regarding how biochemical signals are transduced into alternatively spliced RNA molecules and provide crucial tools for discovering the function of circRNA. Specifically, we aim to 1) engineer statistical algorithms for detecting and quantifying circRNA variants, and statistical methods for integrating expression across datasets; 2) discover trans-acting factors regulating circRNA production, export and decay; 3) systematically discover cis sequence control of circRNA abundance. The work will build on our discoveries of regulated expression of circular in human development, to delineate their regulation under normal circumstances, and how dysregulation may contribute to diseases such as neurodegeneration and cardiomyopathy.